Methods of detecting silencing mammalian cells

ABSTRACT

Methods are disclosed for screening of the occurrence of gene silencing (e.g. post transcriptional gene silencing) in an organism. Also provided are methods for isolating silencing agents so identified.

This application is a divisional application of U.S. application Ser.No. 09/491,549, filed Jan. 26, 2000 now U.S. Pat. No. 6,753,139, whichin turn claims priority to GB 9925459.1, filed Oct. 27, 1999. Each ofthe above identified applications is incorporated by reference herein.

TECHNICAL FIELD

The present invention relates generally to methods and materials for usein achieving and detecting gene silencing, particularlypost-transcriptional gene silencing, in an organism.

PRIOR ART

Methods of detecting and efficiently achieving gene silencing are ofgreat interest to those skilled in the art.

Post-transcriptional gene silencing (PTGS) is a nucleotidesequence-specific defence mechanism that can target both cellular andviral mRNAs. PTGS occurs in plants and fungi transformed with foreign orendogenous DNA and results in the reduced accumulation of RNA moleculeswith sequence similarity to the introduced nucleic acid (1, 2).

PTGS in plants can be suppressed by several virus-encoded proteins (6)and is closely related to RNA-mediated virus resistance andcross-protection in plants (7,8). Therefore, PTGS may represent anatural antiviral defence mechanism and transgenes might be targetedbecause they, or their RNA, are perceived as viruses. PTGS could alsorepresents a defence system against transposable elements and mayfunction in plant development (9-11). To account for the sequencespecificity, and post-transcriptional nature of PTGS it has beenproposed that antisense RNA forms a duplex with the target RNA therebypromoting its degradation or interfering with its translation (12).

One problem which exists in actually utilising efficient gene silencing,for instance via anti-sense mechanisms, is selecting appropriate regionsto target. This problem has been reviewed in the literature (see Szoka(1997) Nature Biotechnology 15: 509; Eckstein (1998) NatureBiotechnology 16: 24). Proposed solutions to selecting good targetregions include computational analysis (Patzel and Sczakiel (1998)Nature Biotechnology 16: 64-68) or Rnase H cleavage using chimericanti-sense oligonucleotides (see Ho (1996) Nucleic Acid Res 24:1901-1907; Ho et al (1998) Nature Biotechnology 16: 59-62). Other groupshave used wide array of oligonucleotides to select those which formheteroduplexes (see Milner et al (1997) Nature Biotechnology 15:537-541).

DISCLOSURE OF THE INVENTION

The present inventors have investigated PTGS of target genes initiatedby a variety of silencing mechanisms in different organisms, and haveestablished that in every case a previously uncharacterised species ofantisense RNA complementary to the targeted mRNA was detected. These RNAmolecules were of a uniform length, estimated at around 25 nucleotides,and their accumulation required either transgene sense transcription orRNA virus replication. Corresponding sense RNA molecules were alsodetected.

There have been no previous reports of such short sense and antisenseRNA molecules (hereinafter, collectively, SRMs) that are detectedexclusively in organisms exhibiting PTGS, possibly because (owing totheir size) they could not have been readily detected by routine RNAanalyses.

It appears that the SRMs may be synthesized from an RNA template andrepresent a specificity determinant and molecular marker of PTGS.Because of their correlation with PTGS and the nature of the molecules(short complementary molecules which could base pair with the targetRNAs) they are believed to represent a signal and/or inducer oractivator of PTGS.

The identification of this species by the present inventors may beutilised by those skilled in the art in a variety of methods andprocesses which are discussed in more detail below. Generally speakingthe present invention provides, inter alia, methods of identifying andscreening for gene silencing and particular silenced genes in organisms;processes for producing or isolating silencing agents, and such isolatedagents themselves; methods for selecting target regions of nucleic acidswhich it is desired to silence and methods for silencing target genesusing the agents or target regions generated as above. Also included arerelevant materials (e.g. nucleic acids, constructs, host cells,transgenic plants, silenced organisms) and methods of use of these.

Importantly, the disclosure herein provides evidence that SRMs may be acommon mediator of PTGS in both plants and higher organisms, such as thenematode discussed in the Examples hereinafter. It was previously knownthat double stranded RNA induces a similar effect to plant PTGS innematodes, insects (4) and protozoa (5). For instance PTGS has beendemonstrated in Caenorhabditis elegans (a nematode worm) using DsRNAintroduced into the worms by microinjection, imbibing or by allowing theworms to eat bacteria (E. coli) which are synthesizing dsRNA. There wasalso some evidence that in some examples of PTGS in plants and dsRNAinterference in nematodes, a signal is produced which spreads andamplifies the silencing beyond the point of introduction of the originalinducer of silencing. Although there were known to be certainsimilarities between the DsRNA induced silencing in nematodes and thecauses of PTGS in plants, there was no clear evidence that the two arerelated.

Aspects of the invention will now be discussed in more detail.

Thus in one aspect of the present invention there is provided a methodof detecting, diagnosing, or screening for gene silencing in anorganism, which method comprises the steps of:

-   (i) obtaining sample material from the organism,-   (ii) extracting nucleic acid material therefrom,-   (iii) analysing the extracted nucleic acid in order to detect the    presence or absence of SRMs therein,

The result of the analysis in step (iii) may be correlated with thepresence of silencing in the organism.

The ‘sample’ may be all or part of the organism, but will include atleast some cellular material.

The term ‘SRMs’ is used to describe the short RNA molecules describedherein which are approximately 25 nucleotides in length. The sizeappears to be very characteristic, being estimated as approximately 25nucleotides in all the cases tested (relative to the same molecular sizemarkers when assessed by chromatography). However, it may be slightlymore or less than this characteristic length (say plus or minus 1, 2, 3,4 or 5 nucleotides) and where the term ‘25 nt RNA’ is used herein, itwill be understood by those skilled in the art that the comments wouldapply equally in the event that the SRMs do not have this preciselength.

Indeed the precise length may not be important, since the disclosureherein permits the identification, isolation and utilisation of SRMS inany case.

In performing the invention, it may be preferred to analyse or otherwiseutilise short anti-sense RNA molecules (SARMs) rather than short senseRNA molecules (SSRMs). Nonetheless, where reference is made herein toSARMs (except where context clearly suggests otherwise) it will beappreciated by those skilled in the art that the SSRMs may also be used.

In particular, the SRMs methodology may be used as an indicator of PTGS.As is well known to those skilled in the art, PTGS occurspost-transcriptionally: i.e. the transcription rates of the suppressedgenes are unaffected. The term ‘gene’ is used broadly to describe anysequence which is suitable for translation to a protein.

Thus the presence of SRMs can be used as a diagnostic test for theexistence of PTGS.

In one embodiment of this aspect there is disclosed a method ofdetecting or identifying the silencing of a target gene in an organism,which method further comprises characterising any SRMs which arepresent. It should be noted that PTGS effects are very dominant. Inprinciple the presence of SRMs may indicate the silencing of more thanone gene, providing that they have sufficient homology.

‘Characterised’ and ‘characterising’ does not necessarily imply completesequencing, although this may be preferred. In order to detect silencingof a known sequence, the SRMs may be fully or partially sequenced, orsequence identity or similarity may be inferred from e.g. blotting.

Applications for such a diagnostic test will depend on the organism inquestion. For instance, in plants, since PTGS is the basis for a lot ofpathogen derived resistance (PDR), GM field crops (e.g. individuals, orpopulations) engineered for PDR could be monitored “in field” bychecking for the existence of 25 nt RNA to make sure that the PDR wasstill operating prior to the attack by the virus.

Similarly, crops depending upon co-suppression for the knockout of aparticular plant gene to achieve a specific modified trait could beassayed for the continued function of PTGS by checking the presence of25 nt RNA against the intended target. Such an assay may be particularlyuseful in view of evidence that transgenes have a tendency to becometranscriptionally inactivated over the generations. PTGS depends upontranscription of the initiating transgene to function and so if thisgets reduced the PTGS will begin to fail. Monitoring 25 nt RNA providesa quick way to test the lines. Non-limiting Examples of silenced geneswhich could be monitored in this way include any of those which havealready been shown to be suppressible by PTGS in the literature. Thesemay include, for example, chalcone synthase of petunia orpolygalacturonase of tomato (Jorgensen, R. A. (1995), Science, 268:686-691, Hamilton, A. J., et al (1995), Current Topics In Microbiologyand Immunology, 197: 77-89).

It is also possible that the process of PTGS underlies certain plantdevelopmental processes. If there are plant genes which are beingtargeted naturally as a result of PTGS in order to satisfy some plantdevelopmental programme, a 25 nt RNA corresponding to sequences fromthese genes may be detectable.

Thus, in this embodiment, the SRMs may be used to identify and isolatean unknown target. This could be achieved by analysing the 25 nucleotidefraction of RNA from a plant, tagging it with a marker (e.g. aradioactive one) and then using this radioactive RNA to probe a libraryof plant genes. This probe will anneal to genes which are undergoingPTGS in the plant, which genes can then be further analysed orcharacterised if required. Such genes, inasmuch as they are novel,represent a further aspect of the present invention.

In a further aspect of the present invention, there is disclosed aprocess for producing or isolating short RNA molecules. As discussedabove, SRMs may not be readily detected by routine RNA analyses,particularly those which include a step in which such molecules are‘lost’ (for instance SRMS may not be efficiently retained on silicacolumns which are used to isolate longer molecules such as mRNAs). Apreferred process is set out in the Examples hereinafter. Broadlyspeaking, the processes provided divide into two parts:extraction/purification and detection.

For extraction, initial steps may be performed using conventional RNAextraction methods and kits appropriate to the organism in question,modified as required to ensure that SRMs are retained at each step.

In order to enhance purification of short RNAs, the extraction mayoptionally be followed by one or more of the following steps:

-   (i) filtration (e.g. through Centricon 100 concentrators (Amicon) or    similar),-   (ii) differential precipitation (e.g. with 5% polyethylene glycol    (8000)/0.5M NaCl)-   (iii) ion exchange chromatography (e.g. using Qiagen columns).

These steps enrich and purify the short RNAs to greater degrees than isobtained with the routine rRNA extraction method alone, and may beperformed in conventional manner using, if required, proprietaryreagents.

It should be noted that there is no requirement that the short RNAs bepurified to homogeneity, provided only that they are capable ofdetection.

Regarding detection, because of their small size the method for this isnot the usual one for “RNA gel blot analysis” although the principle isthe same i.e. separation of the RNA molecules according to size byelectrophoresis through a gel.

Preferably the gel is a 15% polyacrylamide gel containing 7M urea as adenaturant and TBE (0.5×) as a buffer.

The RNAs are preferably transferred to a hybridisation membrane byelectrophoresis (rather than the more conventional capillary blot). Oncethe RNA is on the membrane, it is covalently attached to it by UVirradiation. The membrane is then placed in “prehybridisation solution”for a short time.

Radioactive probe may be prepared using standard techniques. However,preferably, it is made as a single stranded RNA molecule transcribed invitro from an appropriate plasmid DNA templates. The length of the probemay, preferably, be shortened by limited hydrolysis before adding to theprehybridisation solution; this may reduce non-sequence specific bindingof probe to the membrane.

The hybridisation of the probe to its target is allowed to proceed at astringency level (specific temperature, salt concentration and theconcentration of formamide in the prehybridisation solution) appropriateto the requirements of the process. For instance low temperature, highsalt, no formamide equals a low stringency, which may permit shortprobes or probes with imperfect homology to the target to hybridise withthe target. Conversely high temperature, low salt and formamide meanhigh stringency with only lengthy duplexes stable under theseconditions. Preferred conditions are 45% formamide, 7% SDS, 0.3M NaCl,0.05M Na₂HPO₄/NaH₂PO₄ (pH 7), 1× Denhardt's solution, and singlestranded heterologous nucleic acid (e.g. derived from salmon sperm).

This is one preferred process of purifying (or partially purifying) SRMsfor the purpose of detection and/or further characterising e.g. forsequencing. However it should be understood that the present inventionis in no way limited to this particular format, and others methods forSRMs analysis, such as those which may be devised in the future, willalso be encompassed.

The process described above may form part of a more extensive processfor producing or isolating a silencing agent for a target gene, whichsilencing agent is a preferably a SRM, the process comprising the stepsof:

-   (i) silencing a target gene in an organism,-   (ii) performing a process as described above in order to isolate a    SRM appropriate for that gene.

‘Silencing agent’ in this context may be one or more of an inducer,signal, or specificity determinant of gene silencing, particularly PTGS.Preferably this will be a SARM (as opposed to a SSRM). Isolatedsilencing agents obtained or obtainable by this method, inasmuch as theyare novel, form a further aspect of the present invention.

The initial silencing step may be achieved by any conventional methodappropriate to the organism in question. For instance in plants it couldbe by silencing of endogenous, homologous genes (co-suppression—see, forexample, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoliet al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The PlantCell 4, 1575-1588, and U.S. Pat. No. 5,231,020). Further refinements ofthe gene silencing or co-suppression technology may be found inWO95/34668 (Biosource); Angell & Baulcombe (1997) The EMBO Journal16,12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553(systemically induced transgene silencing). Other options includetransgene silencing; RNA mediated defence against viral infection, andtransgenic, homology-dependent, virus resistance, or use of dsRNA in thecase of nematodes.

In a further aspect of the present invention there is disclosed a methodfor identifying or selecting a target region of a gene, which gene it isdesired to silence, which method comprises:

-   (i) silencing the target gene in an organism,-   (ii) performing a process as described above in order to isolate a    SRM appropriate for that gene,-   (iii) identifying a region in the sequence of the gene which    corresponds to the sequence of the SRM.

The region may identified most readily by comparing the sequence of theSRM with the sequence of the gene; however any appropriate method may beused (e.g. RNAase protection). If several SRMs are isolated, thenseveral target regions may be identified.

As described in the introduction, this method provides an alternative toe.g. computational analysis in order to identify the most suitable siteon e.g. an mRNA corresponding to a target gene, for targeting forsilencing e.g. with an anti-sense construct. With the informationobtained using the methods and processes herein about, more efficientantisense reagents (not necessarily RNAs) may be produced which aretailored such that they would be recognised and used by the PTGSmachinery of the organism.

In a further aspect of the present invention there is disclosed a methodof silencing a target gene in an organism which utilises the methodologydescribed above.

“Silencing” in this context is a term generally used to refer tosuppression of expression of a gene. The degree of reduction may be soas to totally abolish production of the encoded gene product, but moreusually the abolition of expression is partial, with some degree ofexpression remaining. The term should not therefore be taken to requirecomplete “silencing” of expression. It is used herein where convenientbecause those skilled in the art well understand this.

In one embodiment, the method comprises introducing anti-sense molecules[SARMs] appropriate for the target gene into the organism in order toinduce silencing. This could be done, for instance, by use oftranscribable constructs encoding the SARMs.

In a related embodiment, the silencing may be achieved using constructstargeting those regions identified by the SRMs-based method disclosedabove. Such constructs may e.g. encode anti-sense oligonucleotides whichtarget all are part of the identified region, or a region within 1, 2,3, 4, 5, 10, 15 or 20 nucleotides of the identified region.

Suitable target genes for silencing will occur to those skilled in theart as appropriate to the problem in hand. For instance, in plants, itmay be desirable to silence genes conferring unwanted traits in theplant by transformation with transgene constructs containing elements ofthese genes. Examples of this type of application include silencing ofripening specific genes in tomato to improve processing and handlingcharacteristics of the harvested fruit; silencing of genes involved inpollen formation so that breeders can reproducibly generate male sterileplants for the production of F1 hybrids; silencing of genes involved inlignin biosynthesis to facilitate paper making from vegetative tissue ofthe plant; silencing of genes involved in flower pigment production toproduce novel flower colours; silencing of genes involved in regulatorypathways controlling development or environmental responses to produceplants with novel growth habit or (for example) disease resistance;elimination of toxic secondary metabolites by silencing of genesrequired for toxin production. In addition, silencing can be useful as ameans of developing virus resistant plants when the transgene is similarto a viral genome.

As described above, the disclosure herein provides evidence that SRMsmay be a common mediator of PTGS in both plants and higher organisms.These new findings can be utilised, inter alia, in that it now appearsthat induction of SRMs (particularly SARMs) with an appropriatespecificity in one organism (say, a plant) may be used to silence anappropriate target gene in another organism (say, a predator) whichcomes into contact with that plant.

In one aspect of the invention there is provided a method for targetinga gene in a first organism, which method comprises generating a SARMssilencing agent in a second organism, and introducing the SARMs into thefirst organism.

The SARMs may be generated by any appropriate silencing method.Preferably the target gene will be one which is not an endogenous genein the second organism (but preferably is endogenous to the first). The‘contact’ may be ingestion, injection, or any other method ofadministration. How, precisely, the method is performed will depend onthe organisms and genes involved.

For instance, in the case of plants and plant predators, it is knownthat the systemic signal of PTGS travels out of plant cells into thephloem (sap) of plants and induces silencing in previously non-silencingparts of the plant. In the light of the present disclosure it is clearthat, since plant parasitic nematodes feed directly upon the sap andcontents of plant cells, they will ingest the signal and inducer of PTGS(i.e. SARMs) in the plant.

As shown in the Examples below, it appears that the same type of SARMsare present in C. elegans which are undergoing PTGS induced by theingestion of dsRNA. This implies that the mechanism of PTGS in plantsand nematode is similar if not identical. Thus plant SARMs may triggerthe PTGS of any similar sequences present in the worm. Therefore whenthe nematode feeds on the plant, and eats the PTGS signal, if there ishomology between the plant's transgene from which the PTGS signalderived and a nematode gene, PTGS of that gene ought to be triggered inthe worm.

Where the targeted gene is an essential gene, this method provides ameans of controlling or killing plant predators or pests. Naturally,more than one gene can be targeted at once.

It may be desirable that the targeted gene is one which is either notpresent, or not important, in the wild-type plant or other potentialconsumers of the plant i.e. is nematode specific gene, such as anematode protease gene. This gives the method a high degree ofspecificity.

Interestingly C. elegans is a nematode distantly related to thenematodes that parasitise plants. Since dsRNA induced PTGS is conservedbetween nematodes, protozoa and insects it is likely that these otherorganisms which support PTGS may be susceptible to SARMs.

DsRNA interference has also been shown to work in insects and transgeneinduced PTGS works in fungi, so it is likely that this is a mechanismthat is broadly conserved across the kingdoms. This implies that anyorganism that directly feeds off plant cellular contents orextracellular components such as sap could ingest PTGS specific SARMS.If these have sequence homology with genes resident in the parasite,PTGS of these genes could be initiated.

Thus insect specific genes (e.g. from aphids) represent a furthertarget. Most preferable would be those insect genes or sequences notfound in beneficial insects, such a ladybirds.

Other targets include genes specific for plant parasites of plants whichfeed off the host plant.

Specifically regarding higher animals (e.g. mammals, fish, birds,reptiles etc.) methods of the present invention include, inter alia:

-   (i) methods for detecting or diagnosing gene silencing, or silencing    of particular genes, in the animal by using SRMs as described above,-   (ii) methods for identifying silenced genes in the animal by using    SRMs as described above,-   (iii) methods for selecting target sites on genes to be silenced    using SRMs as described above,-   (iv) method for silencing a target gene in the animal, either    directly, or through an animal-derived transgene in a second    organism (e.g. a plant) as described above.

Generally speaking target genes in animals may be those whose functionalimpairment beings therapeutic benefits. Typical genes of interest may be(for instance) those involved apoptosis, cancer, cell-cycle regulation,neurological processes, signal transduction etc. Examples and referencescan be found in the Oncogene Research Products 1999 General Catalog, pp21-265, available from Oncogene Research Products, 84 Rogers Street,Cambridge, Mass. 02142, U.S. Preferred examples include oncogenes,transcriptional regulators, pocket proteins, members of the MHCsuperfamily (to produce allotypic organs) etc.

Some further aspects and applications for the present invention will nowbe discussed.

According to one aspect of the present invention there is provided,preferably within a vector suitable for stable transformation of a plantcell, a DNA construct in which a promoter is operably linked to DNA fortranscription in a plant cell to generate either:

-   (i) a SARM as described above, or-   (ii) an anti-sense RNA molecule selected to target a region    identified by the SRM-based methods discussed above.

Generally speaking, such constructs may be used to silence genes withinplants, or within organisms predating or being administered materialfrom plants, in the terms discussed above. Anti-sense partial genesequences selected in accordance with SRM-based methods may be usedanalogously to those previously used in the art. See, for example,Rothstein et al, 1987; Smith et al, (1988) Nature 334, 724-726; Zhang etal, (1992) The Plant Cell 4, 1575-1588, English et al., (1996) The PlantCell 8, 179-188. Antisense technology is also reviewed in Bourque,(1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91,3490-3496. Generally the selected sequence will be less than 50, 40, 30,25, or 20 nucleotides. It may be preferable that there is completesequence identity in the targeting (e.g. foreign) sequence in theconstruct and the target sequence in the plant, although totalcomplementarity or similarity of sequence is not essential.

Again, generally speaking, plants and associated methods and processeswhich form a part of the present invention are either those which:

-   (i) are transformed with the ‘targeting’ anti-sense vectors such as    those described above, for instance so as to silence an (endogenous)    target gene in the plant or perhaps a viral gene, or-   (ii) are transformed with transgenes taken from other organisms such    as to induce transgene silencing and thereby generate SARMs which    can be used to silence a target gene in that other organism, or-   (iii) are transformed with vectors which encode SARMs directly,    which can be used for either purpose.

The general methodology discussed below will be applicable to all ofthese applications.

A vector which contains the construct may be used in transformation ofone or more plant cells to introduce the construct stably into thegenome, so that it is stably inherited from one generation to the next.This is preferably followed by regeneration of a plant from such cellsto produce a transgenic plant. Thus, in further aspects, the presentinvention also provides the use of the construct or vector in productionof a transgenic plant, methods of transformation of cells and plants,plant and microbial (particularly Agrobacterium) cells, and variousplant products.

The function of the promoter in the construct is to ensure that the DNAis transcribed into RNA containing the viral sequences. By “promoter” ismeant a sequence of nucleotides from which transcription may beinitiated of DNA operably linked downstream (i.e. in the 3′ direction onthe sense strand of double-stranded DNA). A promoter “drives”transcription of an operably linked sequence.

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter.

Preferred promoters may include the 35S promoter of cauliflower mosaicvirus or the nopaline synthase promoter of Agrobacterium tumefaciens(Sanders, P. R., et al (1987), Nucleic Acids Res., 15: 1543-1558). Thesepromoters are expressed in many, if not all, cell types of many plants.Depending on the target gene of amplicon gs, other promoters includingthose that are developmentally regulated or inducible may be used. Forexample, if it is necessary to silence the target gene specifically in aparticular cell type the construct may be assembled with a promoter thatdrives transcription only in that cell type. Similarly, if the targetgene is to be silenced following a defined external stimulus theconstruct may incorporate a promoter that is be activated specificallyby that stimulus. Promoters that are both tissue specific and inducibleby specific stimuli may be used. Suitable promoters may include themaize glutathione-S-transferase isoform II (GST-II-27) gene promoterwhich is activated in response to application of exogenous safener(WO93/01294, ICI Ltd).

An additional optional feature of a construct used in accordance withthe present invention is a transcriptional terminator. Thetranscriptional terminator from nopaline synthase gene of agrobacteriumtumefaciens (Depicker, A., et al (1982), J. Mol. Appl. Genet., 1:561-573) may be used. Other suitable transcriptional terminators will bewell known to those skilled in the art.

Those skilled in the art are well able to construct vectors (includingthose based on ‘naked’ DNA) and design protocols for recombinant geneexpression. For further details see, for example, Molecular Cloning: aLaboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring HarborLaboratory Press. Many known techniques and protocols for manipulationof nucleic acid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail inProtocols in Molecular Biology, Second Edition, Ausubel et al. eds.,John Wiley & Sons, 1992.

Specific procedures and vectors previously used with wide success uponplants are described by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721),and Guerineau and Mullineaux, (1993) Plant transformation and expressionvectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOSScientific Publishers, pp 121-148.

For introduction into a plant cell, the nucleic acid construct may be inthe form of a recombinant vector, for example an Agrobacterium binaryvector. Microbial, particularly bacterial and especially Agrobacterium,host cells containing a construct according to the invention or a vectorwhich includes such a construct, particularly a binary vector suitablefor stable transformation of a plant cell, are also provided by thepresent invention.

Nucleic acid molecules, constructs and vectors according to the presentinvention may be provided isolated and/or purified (i.e. from theirnatural environment), in substantially pure or homogeneous form, or freeor substantially free of other nucleic acid. Nucleic acid according tothe present invention may be wholly or partially synthetic. The term“isolate” encompasses all these possibilities.

An aspect of the present invention is the use of a construct or vectoraccording to the invention in the production of a transgenic plant.

A further aspect provides a method including introducing the constructor vector into a plant cell such that the construct is stablyincorporated into the genome of the cell.

Any appropriate method of plant transformation may be used to generateplant cells containing a construct within the genome in accordance withthe present invention. Following transformation, plants may beregenerated from transformed plant cells and tissue.

Successfully transformed cells and/or plants, i.e. with the constructincorporated into their genome, may be selected following introductionof the nucleic acid into plant cells, optionally followed byregeneration into a plant, e.g. using one or more marker genes such asantibiotic resistance. Selectable genetic markers may be used consistingof chimeric genes that confer selectable phenotypes such as resistanceto antibiotics such as kanamycin, hygromycin, phosphinotricin,chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinonesand glyphosate.

When introducing a nucleic acid into a cell, certain considerations mustbe taken into account, well known to those skilled in the art. Thenucleic acid to be inserted should be assembled within a construct whichcontains effective regulatory elements which will drive transcription.There must be available a method of transporting the construct into thecell. Once the construct is within the cell membrane, integration intothe endogenous chromosomal material should occur. Finally, as far asplants are concerned the target cell type must be such that cells can beregenerated into whole plants.

Plants transformed with the DNA segment containing the sequence may beproduced by standard techniques which are already known for the geneticmanipulation of plants. DNA can be transformed into plant cells usingany suitable technology, such as a disarmed Ti-plasmid vector carried byAgrobacterium exploiting its natural gene transfer ability (EP-A-270355,EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectilebombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616)microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green etal. (1987) Plant Tissue and Cell Culture, Academic Press),electroporation (EP 290395; WO 8706614 Gelvin Debeyser—see attached)other forms of direct DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No.4,684,611), liposome mediated DNA uptake (e.g. Freeman et al. Plant CellPhysiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNASU.S.A. 87: 1228 (1990d). Physical methods for the transformation ofplant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al.(1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al.(1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology9, 957-962; Peng, et al. (1991) International Rice Research Institute,Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11,585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al.(1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990)Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2,603-618; D'Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, etal. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993)Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084;Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828). Inparticular, Agrobacterium mediated transformation is now emerging alsoas an highly efficient transformation method in monocots (Hiei et al.(1994) The Plant Journal 6, 271-282).

The generation of fertile transgenic plants has been achieved in thecereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K.(1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al.(1992) Bio/Technology 10, 667-674; Vain et al., 1995, BiotechnologyAdvances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page702).

Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, e.g. bombardmentwith Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

Following transformation, a plant may be regenerated, e.g. from singlecells, callus tissue or leaf discs, as is standard in the art. Almostany plant can be entirely regenerated from cells, tissues and organs ofthe plant. Available techniques are reviewed in Vasil et al., CellCulture and Somatic Cel Genetics of Plants, Vol I, II and III,Laboratory Procedures and Their Applications, Academic Press, 1984, andWeissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989.

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practicing the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

Also according to the invention there is provided a plant cell havingincorporated into its genome a DNA construct as disclosed. A furtheraspect of the present invention provides a method of making such a plantcell involving introduction of a vector including the construct into aplant cell. Such introduction should be followed by recombinationbetween the vector and the plant cell genome to introduce the sequenceof nucleotides into the genome. RNA encoded by the introduced nucleicacid construct may then be transcribed in the cell and descendantsthereof, including cells in plants regenerated from transformedmaterial. A gene stably incorporated into the genome of a plant ispassed from generation to generation to descendants of the plant, sosuch descendants should show the desired phenotype.

The present invention also provides a plant comprising a plant cell asdisclosed.

A plant according to the present invention may be one which does notbreed true in one or more properties. Plant varieties may be excluded,particularly registrable plant varieties according to Plant Breeders'Rights.

In addition to a plant, the present invention provides any clone of sucha plant, seed, selfed or hybrid progeny and descendants, and any part ofany of these, such as cuttings, seed.

The present invention may be used in plants such as crop plants,including cereals and pulses, maize, wheat, potatoes, tapioca, rice,sorgum, millet, cassaya, barley, pea and other root, tuber or seedcrops. Important seed crops are oil seed rape, sugar beet, maize,sunflower, soybean and sorghum. Horticultural plants to which thepresent invention may be applied may include lettuce, endive andvegetable brassicas including cabbage, broccoli and cauliflower, andcarnations and geraniums. The present invention may be applied totobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper,chrysanthemum, poplar, eucalyptus and pine.

In relation to use in mammals or other higher animals, DNA vectors(including naked DNA suitable for expression in mammals) of the presentinvention encode either:

-   (i) a SARM as described above, or-   (ii) an anti-sense RNA molecule selected to target a region    identified by the SRM-based methods discussed above.

Such vector may be based on any appropriate vector known to thoseskilled in the art. For instance incorporation of this DNA intomammalian cells to produce such antisense RNA in vivo might beaccomplished using vectors based on the disclosure of European patentapplication 909052736.3 (VICAL), HSV, vaccinia or adenovirus (seePrinciples of Gene Manipulation (1994) 5th Edit. Old and Primrose 5thEdition, Blackwell Scientific Publications). Viral vectors for use ingene therapy are discussed by Vile (1997) Nature Biotechnology 15:840-841. A non-viral gene therapy approach is discussed by Sebestyen etal (1998) Nature Biotechnology 16: 80-85. The use of a variety of genetherapy delivery systems (including HSV VP22) is discussed by Fernandez& Baylay (1998) Nature Biotechnology 16: 418-420 and references therein.

Also provided by the present invention is an organism, preferably anon-human mammal, comprising cells in which a target gene is subject toPTGS by use of the SARM-based methods or materials disclosed herein.Particularly preferred is a rodent e.g. murine organism. In thisembodiment the invention provides an alternative to known methods ofproducing ‘knock out’ mammals in which specific gene activities havebeen impaired (see e.g. Boerrigter et al (1995) Nature 377: 657-659, orGossen and Vijk (1993) Trends Genet 9: 27-31.)

The invention will now be further described with reference to thefollowing non-limiting Examples describing work of the inventors. Theresults are also discussed, and suggestions made as to the origin of theSRMs of the present invention. However it will be appreciated by thoseskilled in the art that the materials, methods and processes in thepresent disclosure may be usefully applied irrespective of the preciseunderlying mechanisms involved.

All references discussed herein, inasmuch as they may be required tosupplement the present disclosure, are incorporated herein by reference.

EXAMPLES Example 1 Detection of SRMs in Silenced Plants

Analyses were performed to detect low molecular weight antisense RNA infour classes of PTGS in plants using the following general methods.

Total RNA was extracted from leaves of tomato, tobacco and N.benthamiana as described previously (E. Mueller, J. E. Gilbert, G.Davenport, G. Brigneti, D. C. Baulcombe, Plant J. 7, 1001 (1995)). Fromthese preparations, low molecular weight RNA was enriched by ionexchange chromatography on Qiagen columns following removal of highmolecular weight species by precipitation with 5% polyethylene glycol(8000)/0.5M NaCl (for tobacco and N. benthamiana) or (for tomato) byfiltration through Centricon 100 concentrators (Amicon). Low molecularweight RNA was separated by electrophoresis through 15%polyacrylamide/7M urea/0.5×TBE gels, transferred onto Hybond Nx filters(Amersham) and fixed by UV crosslinking. Prehybridization was in 45%formamide, 7% SDS, 0.3M NaCl, 0.05M Na₂HPO₄/NaH₂PO₄ (pH 7), 1×Denhardt's solution, 100 mg.ml.⁻¹ sheared, denatured, salmon sperm DNAat between 30° C. and 40° C. Hybridization was in the same solution withsingle stranded RNA probes transcribed with a-³²P-labelled UTP. Beforeaddition to the filters in the prehybridization solution, probes werehydrolysed to lengths averaging 50 nucleotides. Hybridization was for 16hours at 30° C. (ACO probes), 35° C. (GUS probe) or 40° C. (GFP and PVXprobes).

Sizes of RNA molecules were estimated by comparison with ³³Pphosphorylated DNA oligonucleotides run on the same gels but imagedseparately. Additionally, samples from different types of PTGS includingthose discussed were frequently run on the same gel. Alignment of thefilters following hybridization with different specific probes confirmedthat the PTGS specific signals were identical in size. The probes usedare in each case sequence specific. We have observed nocross-hybridization between 25 nt signals in different PTGS systemsusing either filter hybridisation or RNAase protection

We do not have an exact measurement of amount of 25 nt per cell, butgiven the short exposure times routinely used to detect these moleculesand taking into account their size, they are likely to be very abundantin cells exhibiting PTGS.

Co-suppression

The first class tested was transgene-induced PTGS of an endogenous gene(“co-suppression”). We used five tomato lines (T1.1, T1.2, T5.1, T5.2,T5.3), each transformed with a tomato 1-aminocyclopropane-1-carboxylateoxidase (ACO) cDNA sequence placed downstream of the cauliflower mosaicvirus 35S promoter (35S). Two lines (T5.2, T5.3) exhibited PTGS of theendogenous ACO mRNA when amplified by RT-PCR and detected byhybridization with labelled ACO cDNA.

Low molecular weight nucleic acids purified from the five lines wereseparated by denaturing polyacrylamide gel electrophoresis, blotted, andhybridized to an ACO sense (antisense-specific) RNA probe. Morespecifically, the low molecular weight RNA and a 30-mer ACO antisenseRNA oligonucleotide were fractionated, blotted and hybridized witheither ACO sense RNA or antisense RNA transcribed from full length ACOcDNA. The low hybridisation temperature permitted some non-specifichybridization to tRNA and small rRNA species which constitute most ofthe RNA mass in these fractions. A discrete, ACO antisense RNA of 25nucleotides (nt) was present in both PTGS lines but absent from thenon-silencing lines. 25 nt ACO RNA of sense polarity and at the sameabundance as the 25 nt ACO antisense RNA was also present only in thePTGS lines. The 25 nt ACO antisense signal was completely abolished bypretreatment with either RNAaseONE or NaOH.

Transgene Silencing

PTGS induced by transgenes can also occur when a transgene does not havehomology to an endogenous gene (1). Therefore we tested whether thistype of PTGS was also associated with small antisense RNA. We analysedthree tobacco lines carrying 35S-b-glucuronidase (GUS) transgenes. Twoof these lines, T4 (15) and 6b5 (16) exhibited PTGS of GUS. The thirdline (6b5×271) tested was produced by crossing 6b5 with line 271 (17) inwhich there is a transgene suppressor of the 35S-promoter in 6b5. Therewas no PTGS of GUS in 6b5×271 due to the transcriptional suppression ofthe 35S GUS transgene (18).

Hybridization with a GUS-specific probe revealed that low molecularweight GUS antisense RNA was present in T4 and 6b5 but absent from line6b5×271. 25 nt GUS antisense RNA was detected by hybridizhybridisationwith hydrolysed GUS sense RNA transcribed from the 3′ 700 bp of the GUScDNA. The amount of antisense RNA correlated with the degree of PTGS:line 6b5 has stronger PTGS of GUS than line T4 (18) and also had moreGUS antisense RNA. It appears that 25 nt antisense GUS RNA is dependentupon transcription from the 35S promoter.

As for PTGS of ACO in tomato, the GUS antisense RNA was a discretespecies of approximately 25 nt.

Systemically Induced Transgene Silencing

In some examples of PTGS, silencing is initiated in a localizsed regionof the plant. A signal molecule is produced at the site of initiationand mediates systemic spread of silencing to other tissues of the plant(19, 20). We investigated whether systemic PTGS of a transgene encodingthe green fluorescent protein (GFP) is associated with 25 nt GFPantisense RNA. PTGS was initiated in Nicotiana benthamiana expressing aGFP transgene by infiltration of a single leaf with Agrobacteriumtumefaciens containing GFP sequences in a binary plant transformationvector.

More specifically, lower leaves of untransformed N. benthamiana and N.benthamiana carrying an active 35S-GFP transgene (35S-GFP) wereinfiltrated with A. tumefaciens containing the same 35S-GFP transgene ina binary vector. Two to three weeks following this infiltration, the GFPfluorescence disappeared due to systemic spread of PTGS as describedpreviously (11, 20).

RNA from upper, non-infiltrated leaves of these plants and fromequivalent leaves of non-infiltrated plants was hybridized with GFPsense RNA transcribed from a full length GFP cDNA. We detected 25 nt GFPantisense RNA in systemic tissues exhibiting PTGS of GFP. It was notdetected in equivalent leaves of plants that had not been infiltrated orin non-transformed plants that had been infiltrated with the A.tumefaciens i.e. only the transgenic N. benthamiana infiltrated with theA. tumefaciens accumulated 25 nt GFP antisense RNA.

RNA-mediated Defence Against Viral Infection

A natural manifestation of PTGS is the RNA-mediated defence induced invirus infected cells (8). Therefore we investigated whethervirus-specific, 25 nt RNA could be detected in a virus-infected plant.

A high titre, synchronised PVX infection on leaves of untransformed N.benthamiana. was initiated by infiltration of single leaves with A.tumefaciens containing a binary plasmid incorporating a 35S-PVX-GFPsequence. Once transcribed, the PVX RNA replicon is independent of the35S-PVX-GFP DNA, replicates to high levels and moves systemicallythrough the plant. The A. tumefaciens does not spread beyond theinfiltrated patch and is not present in systemic leaves (20). The GFPreporter in the virus was used to allow visual monitoring of infectionprogress. We have obtained similar signals with wild type PVX inoculatedas virions in sap taken from an infected plant. RNA was extracted frominoculated leaves after 2, 4, 6 and 10 days and from systemic leavesafter 6 and 10 days. RNA was extracted from mock inoculated leaves after2 days. 25 nt PVX antisense RNA was detected by hybridization with PVXsense RNA transcribed from a full length PVX cDNA. 25 nt RNAcomplementary to the positive strand (genomic) of potato virus X (PVX)was detected 4 days after inoculation of N. benthamiana and continued toaccumulate for at least another 8 days in the inoculated leaf. 25 nt PVXRNA but was not detected in mock inoculated leaves.

Discussion

Thus, 25 nt antisense RNA, complementary to targeted mRNAs, accumulatesin four types of PTGS. We have also detected 25 nt RNA in other examplesof PTGS as follows: N. benthamiana (spontaneous silencing of a 35S-GFPtransgene), tomato (35S-ACO containing an internal direct and invertedrepeat), petunia (co-suppression of chalcone synthase transgenes andendogenes) and Arabidopsis thaliana (PTGS of 35S-GFP by a 35S-PVX-GFPtransgene).

However the 25 nt RNA has never been detected in the absence of PTGS.This correlation and the properties of 25 nt RNA are consistent with adirect role for them in PTGS induced by, for instance, transgenes orviruses (12). 25 nt RNA species also serve as molecular markers forPTGS. Their presence could be used to confirm other examples of e.g.transgene or virus-induced PTGS and may also serve to identifyendogenous genes that are targeted by PTGS in non-transgenic plants. The25 nt antisense RNA species are not degradation products of the targetRNA because they have antisense polarity. A more likely source of theseRNAs is the transcription of an RNA template. This is consistent withthe presence of the 25 nt PVX RNA in PVX infected cells that do notcontain a DNA template. In a further experiment, low molecular weightRNA was extracted from plants containing silencing (S) or non-silencing(NS), 35S-ACC-oxidase (ACO, tomato) or 35S-GFP (N. benthamiana)transgenes. Each was hybridised with ³²P-labelled RNA probes transcribedin the sense orientation from ACC-oxidase and GFP cDNAs and singlestranded RNA then removed by digestion with RNAaseONE (Promega). Theremaining protected RNA molecules were denatured, separated byelectrophoresis on a 15% polyacrylamide/7M urea.0.5×TBE gel. The gel wasdried and imaged by autoradiography. “+” and “−” consist of each probeincubated alone with or without subsequent digestion with RNAaseONE.With the ACO probe, protected fragments are obtained only with RNA fromthe ACO silencing tomato plants and with the GFP probe only with RNAfrom the GFP silencing plants illustrating the sequence specificity ofthe signal. The short RNA species detected in this assay correspond tothe 25 nt RNA detected by northern analysis but are more dispersebecause of RNAase digestion at the ends of breathing RNA duplexes. Somehigher molecular weight signals were also obtained, possible as a resultof incomplete digestion of single stranded regions.

The dependency of 25 nt GUS antisense RNA accumulation on sensetranscription of a GUS transgene also supports the RNA template model.An RNA-dependent RNA polymerase, as required by this model, is requiredfor PTGS in Neurospora crassa (23). With the present data, we cannotdistinguish whether the antisense RNA is made directly as 25 nt speciesor as longer molecules that are subsequently processed. The precise roleof 25 nt RNA in PTGS remains to be determined conclusively. However, asthey are long enough to convey sequence specificity yet small enough tomove through plasmodesmata, it is probable that they are components ofthe systemic signal and specificity determinants of PTGS.

Example 2 Detection of SRMs in Silenced Nematodes

RNA from Caenorhabditis elegans was obtained from Department ofEmbryology, Carnegie Institution of Washington, 115 West UniversityParkway, Baltimore, Md. 21210, USA). RNA was extracted by standardmethods known in the art and was concentrated by ethanol precipitationand redissolved in formamide prior to analysis here.

Nematodes were selected which showed either PTGS (by ingestion of E.coli which synthesise double stranded GFP RNA) or non-silencing of a GFPtransgene.

Northern analysis of this RNA was performed generally as describedabove. RNA was fractionated by electrophoresis through a 15%polyacrylamide gel containing 7M urea and 0.5× Tris Borate EDTA bufferand electrophoretically transferred onto a Hybond Nx filter (Amersham)The membrane was placed on three layers of 3 MM (Whatman) filter papersaturated with 21×SSC for 20 minutes and then allowed to dry at roomtemperature. The RNA was covalently linked to the membrane byUltraviolet radiation crosslinking (“autocrosslink” setting in“Stratalinker” apparatus (Stratagene). The membrane was prehybridized45% formamide, 7% SDS, 0.3M NaCl, 0.05M Na₂HPO₄/NaH₂PO₄ (pH 7), 1×Denhardt's solution, 100 mg.ml.⁻¹ sheared, denatured, salmon sperm DNAat 40° C. Hybridization was in the same solution with a single strandedRNA probe transcribed in the sense orientation with α-³²P-labelled UTPfrom the entire coding sequence of GFP. Before addition to the filter inthe prehybridization solution, the probe was hydrolysed to lengthsaveraging approximately 50 nucleotides by incubation in 100 mMNa₂HCO₃/NaH₂CO₃ (pH 10.2) at 60° C. for 3 hours. Hybridization was for16 hours 40° C. The membrane was washed at 50° C. in 2×SSC/0.2% SDS andthe radioactive signal imaged by a phosphorimager.

As in the previous example, 25 nt. anti-sense RNA was detectable in thesilenced material.

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1. A method of detecting gene silencing of a target gene in a mammalianorganism or in cellular material of a mammalian organism which methodcomprises the steps of: detecting in a nucleic acid extract preparedfrom said organism or in cellular material from said organism thepresence as opposed to the absence of short RNA molecules (SRMs) ofuniform length which are 20-30 nucleotides in length in said extract,characterizing any SRMs which are present in said extract wherein saidcharacterizing comprises determining sequence identity or similaritywith said target gene, wherein the presence of any SRMs having sequenceidentity or similarity with said target gene indicates silencing of saidtarget gene in the organism or in cellular material from said organism,and confirming that said target gene has been silenced.
 2. The method ofclaim 1, wherein the SRMs are short antisense RNA molecules (SARMs). 3.The method of claim 1, wherein the SRMs are short sense RNA molecules(SSRMs).
 4. The method of claim 1, wherein the step of characterizingany SRMs present in the extract to determine sequence identity orsimilarity with a target gene is performed by a process that comprises:tagging said SRMs with a marker, probing a library of genes from saidorganism, and identifying the genes in said library that bind to saidSRMs whereby a gene that binds to said SRM is identified as said targetgene which is silenced.
 5. The method of claim 1, wherein said short RNAmolecules are 20-25 nucleotides in length.
 6. The method of claim 1,wherein the SRMs comprise both short antisense RNA molecules (SARMs) andshort sense RNA molecules (SSRMs).
 7. The method of claim 1, whereinsaid characterizing comprises determining sequence identity with thetarget.
 8. The method of claim 1, wherein as a result of saidcharacterizing, sequences of SRMs are correlated with the occurrence ofgene silencing in said mammalian organism or in cellular material fromsaid organism.
 9. The method of claim 1, wherein said SRMs are 20nucleotides in length.
 10. The method of claim 1, wherein said SRMs are21 nucleotides in length.
 11. The method of claim 1, wherein said SRMsare 22 nucleotides in length.
 12. The method of claim 1, wherein saidSRMs are 23 nucleotides in length.
 13. The method of claim 1, whereinsaid SRMs are 24 nucleotides in length.
 14. The method of claim 1,wherein said SRMs are 25 nucleotides in length.
 15. The method of claim1, wherein said SRMs are 26 nucleotides in length.
 16. The method ofclaim 1, wherein said SRMs are 27 nucleotides in length.
 17. The methodof claim 1, wherein said SRMs are 28 nucleotides in length.
 18. Themethod of claim 1, wherein said SRMs are 29 nucleotides in length. 19.The method of claim 1, wherein said SRMs are 30 nucleotides in length.